Overexpression of ß-Ketoacyl Co-A Synthase1 Gene Improves Tolerance of Drought Susceptible Groundnut (Arachis hypogaea L.) Cultivar K-6 by Increased Leaf Epicuticular Wax Accumulation

Abstract

Drought is one of the major environmental constraints affecting the crop productivity worldwide. One of the agricultural challenges today is to develop plants with minimized water utilization and reduced water loss in adverse environmental conditions. Epicuticular waxes play a major role in minimizing water loss. Epicuticular wax covers aerial plant parts and also prevents non-stomatal water loss by forming the outermost barrier from the surfaces. Epicuticular wax content (EWC) variation was found to be affiliated with drought tolerance of groundnut cultivars. In the current study, a fatty acid elongase gene, KCS1, which catalyzes a rate limiting step in the epicuticular wax biosynthesis was isolated from drought tolerant cultivar K-9 and overexpressed in drought sensitive groundnut cultivar (K-6) under the control of CaMV35S constitutive promoter. Transgenic groundnut plants overexpressing AhKCS1 exhibited normal growth and displaying greenish dark shiny appearance. Environmental scanning electron microscopy (ESEM) revealed the excess of epicuticular wax crystal depositions on the transgenic plant leaves compared to non-transgenic wild type plants. The findings were further supported by gas chromotography-mass spectroscopic analysis (GC-MS) that revealed enhanced levels of fatty acids, secondary alcohols, primary alcohols, aldehydes, alkanes, and ketones in transgenics compared to wild types. The AhKCS1 overexpressing transgenic groundnut plants exhibited increase in the cuticular wax content, reduction of water loss, lower membrane damage, decreased MDA content, and high proline content compared to that of non-transgenic groundnut plants. Our findings suggest that the AhKCS1 gene plays a major role in combating drought stress by preventing non-stomatal water loss in drought sensitive groundnut cultivar (K-6).

Keywords: AhKCS1; drought stress; epicuticular wax; groundnut; non-stomatal water loss; transgenic plants.

FIGURE 1

Analysis of wax crystal morphology of groundnut transgenic, mock, and wild type plants using Environmental Scanning Electron Microscope (QUANTA 250 ESEM). ESEM image showing the variation in epicuticular wax deposition on the leaf surfaces of AhKCSl groundnut transgenic lines 11D-L3, 28D-L1, mock (empty vector), Cultivar K-9, and wild type under drought stress with a magnification of 2000× at 40 μm bar scale.

FIGURE 2

Analyses of leaf wax components in AhKCSl transgenic, mock (empty vector) and wild-type groundnut cultivar K6 by GC-MS. (A) Leaf wax components in AhKCSl transgenic lines 11D-L3 and 28D-L1, mock (empty vector) and wild type, groundnut cultivar K6 showing higher alcohols, alkanes, ketols, ketones, and esters in leaves. (B) Percent increase of wax compounds in two AhKCSl transgenic groundnut leaf samples 11D-L3, 28D-L1, and mock when compared to wild type. The results show average of three replicates and error bars indicate mean ± SE.

FIGURE 3

Drought stress analysis of AhKCSl transgenic groundnut lines, mock, Cultivar K-9, and wild type plants. AhKCSl transgenic groundnut lines showing stay green nature, along with cultivar K-9 and visible wilting symptoms in mock and wild type plants 10 days after drought stress. WT-wild type, M-mock, 11D-L3, and 28D-L1 – AhKCSl transgenic groundnut lines.

FIGURE 4

AhKCSl transgenic groundnut lines 11D-L3, 28D-L1, mock, Cultivar K-9, and wild-type one-month-old plants subjected to 10 days of drought stress and the leaves were collected to estimate different bio-chemical parameters. (A) Estimation of Epicuticular wax content showing increased accumulation of EWC in transgenic lines, onpar with Cultivar K-9. (B) Chlorophyll leaching assay, at different time points, showing the % of chlorophyll extracted was very less in transgenic lines, a little less than that of Cultivar K-9. (C) Rate of water loss was low in transgenic lines, Cultivar K-9 compared to mock and wild type. (D) Estimation of Moisture Retention Capacity revealed that transgenic lines retrained more water in the harvested leaves, along with Cultivar K-9. Values shown are the mean of three replicates and ±SE of three replicates and letters shown above the bars are significantly different at P < 0.05.

FIGURE 5

Leaf material was collected from one month old transgenic groundnut lines 11D-L3, 28D-L1, mock, Cultivar K-9, and wild-type plants subjected to 10 days of drought stress to estimate different bio-chemical parameters. (A) Malondialdehyde (MDA) content was low in transgenic lines and Cultivar K-9. (B) Cell membrane stability (CMS) in transgenic lines and Cultivar K-9 was high with low electrolyte leakage. (C) Relative water content % (RWC) was less in transgenic lines and Cultivar K-9. (D) Proline content was high in transgenic lines and Cultivar K-9. Values shown are the mean of three replicates and ±SE of three replicates and letters shown above the bars are significantly different at P<0.05.